Method of inhibiting hepatitis B virus

ABSTRACT

A method is disclosed for the treatment of hepatitis B virus (HBV) infections which comprises administering to the infected host an N-alkyl derivative of 1,5-dideoxy-1,5-imino-D-glucitol in which the alkyl group contains from 3 to 6 carbon atoms.

CROSS-REFERENCE TO RELATED APPLICATION

This is a 371 OF PCT/US94/14548 filed Dec. 23, 1994 which is acontinuation-in-part of application Ser. No. 08/181,519, filed Jan. 13,1994 now abandoned.

BACKGROUND OF THE INVENTION

This invention relates to a novel method of inhibiting hepatitis B virusand, more particularly, to the use of N-alkyl derivatives of1,5-dideoxy-1,5-imino-D-glucitol for inhibiting replication andsecretion of hepatitis B virus in cells infected with said virus.

Hepatitis B Virus (HBV) is a causative agent of acute and chronic liverdisease [Ayoola et al., Bull. World Health Organ. 66, 443-455 (1988)].Although effective vaccination is available [two HBV vaccines currentlyavailable are Merck's Recombivax HB and SmithKline Beecham's Engerix-B],there are still more than 300 million people worldwide chronicallyinfected with the virus [Eder et al., in Progress in Liver Diseases,eds. Popper and Schaffner (Grune & Stratton, Orlando, FL), vol. 8, pp.367-394 (1986)]. For them, the vaccine has no therapeutic value.According to Dr. Richard Duma, executive director of the NationalFoundation for Infectious Diseases, an estimated 300,000 cases of HBVinfection occur annually in the United States alone [Med. World News34(8), 20-21 (1993)]. Between 25 to 40% of those who are chronicallyinfected with HBV develop serious liver disease. It is thereforeimportant to find effective anti-HBV therapies.

Alpha interferon has been used for treatment of HBV infection withpromising results in some patients [Hoofnagle and Jones, Seminars inLiver Disease 9, 231-233 (1989); and Perrillo, Seminars in Liver Disease9, 240-248 (1989)]. The only treatment for chronic HBV infectioncurrently approved by the U.S. FDA is recombinant interferon alfa-2b(Intron A, Schering-Plough). Clinical tests on the use of the nucleosideanalog, fialuridine, for treatment of chronic hepatitis B were suspendedrecently due to drug-related liver failure in six of 20 patients.Consequently, there is a great need for a safe drug treatment ofhepatitis B.

Recent reports suggest that the virus encoded DNA polymerase, whichfunctions as a reverse transcriptase, is an attractive target [Doong etal., Proc. Natl. Acad. Sci. USA 88, 8495-8499 (1991); Lee et al.,Antimicrob. Aaent Chem. 33, 336-339 (1989); Price et al., Proc. Natl.Acad. Sci. USA 86, 8541-8544 (1989); and Venkateswaran et al., Proc.Natl. Acad. Sci. USA 84, 274-278 (1987)].

Other virus-mediated processes have not been targeted for anti-viralintervention. Effective antiviral therapy for HBV is likely to involvemultiple strategies, including agents that influence the host immunesystem as well as those that interfere with different steps in the lifecycle of the virus. It is therefore of interest to explore thepossibility that other, non-polymerase mediated steps in the virus lifecycle are vulnerable to intervention.

1,5-Dideoxy-1,5-imino-D-glucitol (which is also known as1-deoxynojirimycin or DNJ) and its N-alkyl derivatives are knowninhibitors of the N-linked oligosaccharide processing enzymes,α-glucosidase I and II. Saunier et al., J. Biol. Chem. 257, 14155-14161(1982); Elbein, Ann. Rev. Biochem. 56, 497-534 (1987). As glucoseanalogs they also have potential to inhibit glucosyl-transferases.Newbrun et al., Arch. Oral Biol. 28, 516-536 (1983); Wang et al.,Tetrahedron Lett. 34, 403-406 (1993). Their inhibitory activity againstthe glucosidases has led to the development of these compounds asantihyperglycemic agents and antiviral agents. See, e.g., PCT Int'l.Appln. WO 87/03903 and U.S. Pat. Nos.: 4,065,562; 4,182,767; 4,533,668;4,639,436; 4,849,430; 4,957,926; 5,011,829; and 5,030,638.

Studies on the effect of inhibitory agents on hepatitis B virus (HBV)have been sparse heretofore due to the lack of permissive cell culturesystems for assay purposes. That is, the inability heretofore toreproduce and productively infect tissue cultures with the virus hasbeen a serious limitation to the discovery of useful anti-HBV agents.

In one study, N-methyl deoxynojirimycin has been reported to inhibit theformation of mouse hepatitis virus (MHV) whereby the appearance of E2 onthe cell surface is delayed. See Repp et al., J. Biol. Chem. 280,15873-15879 (1985); Datema et al., Pharmac. Ther. 33, 221-286, at 260(1987). However, MHV is unrelated to the hepatitis B virus (HBV). On theone hand, HBV is a member of the Hepadnavirus family and is a smallvirus pathogen in humans. The HBV size is approximately 42 nM with a DNAgenome size of 3.5 kb.

On the other hand, MHV is a member of the Coronavirus family, and is alarge RNA-containing virus that is not pathogenic to humans, althoughhuman coronavirus pathogens that cause upper respiratory tractinfections are common. The MHV size is about 100-150 nM (being ratherpleiotropic), with an RNA genome size of approximately 30 kb. There arevery few similarities between HBV and MHV. Further backgroundinformation and a complete description of Coronaviruses (including MHV)can be had by reference to K. Holmes, in Virology, 2d edition, ed. by B.Fields, pp. 841-856, Raven Press, New York, N.Y., 1990.

The inability to predict the results from one virus to another isevident from the recent reports by two different scientific groups thathepatitis delta virus (HDV) secretion was not dependent upon HBV sAgglycosylation. W. Hui-Lin et al., Abstr. 115, and C. Gureau et al.,Abstr. 117, in Abstracts of Papers Presented at the 1994 Meeting,"Molecular Biology of Hepatitis B Viruses," Oct. 3-6, 1994, InstitutPasteur, Paris, France.

HDV does not specify its own envelope protein. It infects the same cellsas HBV, and uses the HBV S antigen (HBV envelope protein) to make theinfectious, mature HDV particle. By way of distinction, HBV secretion isdependent upon glycosylation and glycan trimming. That is, although HBVand HDV are composed of the same envelope proteins, HDV secretion isglycosylation independent whereas HBV is very sensitive toglycosylation.

The effect of the glycosylation inhibitor, tunicamycin, on hepatitis Bvirus cell culture systems has been described by Pizer et al., J. Virol.34, 134-153 (1980); Datema et al., supra at 270. However, tunicamycinundesirably and completely prevents the addition of N-linkedoligosaccharides to newly synthesized polypeptide. That is, treatmentwith tunicamycin results in complete inhibition of N-linkedglycosylation of proteins and is very toxic to cells. Moreover,tunicamycin treatment of HBV infected cells resulted in no significantreduction of HBV secretion.

BRIEF DESCRIPTION OF THE INVENTION

In accordance with the present invention a method is provided forinhibiting hepatitis B virus (HBV) in cells infected with said virus.The method comprises treatment of said cells with an N-alkyl derivativeof 1,5-dideoxy-1,5-imino-D-glucitol in which said alkyl group containsfrom 3 to 6 carbon atoms in an effective amount to inhibit replicationand secretion of HBV virions. The N-alkyl group preferably is butyl.

In a preferred illustrative example of the invention,N-butyl-1,5-dideoxy-1,5-imino-D-glucitol (NB-DNJ) is shown to suppressthe secretion of HBV particles and to cause intracellular retention ofHBV DNA in both stably transfected HepG 2.2.15 cells and HBV infectedHepG2 cells.

HepG2 cells are well-known, widely distributed, and readily availablehuman hepatoma cells. The establishment and characteristics of the HepG2cell line are described in U.S. Pat. No. 4,393,133. Samples of this cellline are also available from the American Type Culture Collection,Rockville, Maryland, under accession number ATCC HB 8065, and from theEuropean Collection of Animal Cell Cultures, Porton Down, UK. Thesecells have been used as a source of various proteins, e.g., tissuefactor inhibitor (TFI), also known as lipoprotein associated coagulationinhibitor (LACI), by Broze and Miletich, Proc. Natl. Acad. Sci. USA 84,1886-1890 (1987), and in U.S. Pat. Nos. 4,996,852, 5,106,833 and5,212,091.

HepG 2.2.15 cells are derivatives of HepG2 cells and are prepared asdescribed by Sells et al., Proc. Nat'l. Acad. Sci. USA 84,1005-1009(1987).

The suppression of the secretion of HBV particles by the method of theinvention was most unexpected since it has been previously reported thattunicamycin treatment of HBV-producing cells resulted in normal HBVparticle secretion. See Grippon et al., Mol. Biol. HBV, San Diego,Calif., Abstract page 67 (1992). It has been found by the presentinventors that secretion of HBV virions but not subviral particles isinhibited by tunicamycin.

The increase in intracellular HBV DNA resulting from treatment by themethod of the invention was also unexpected.

Since HBV envelope proteins contain only one or two N-linked glycans permolecule, the ability to inhibit secretion of such modestly (by weight)glycosylated proteins and their virion products by the N-alkylderivatives of DNJ was surprising when contrasted to their lesser effectupon the secretion of HIV which contains heavily glycosylated envelopeproteins. It was also unexpected to find that HBV virions and particlescontaining large cocarboxy- terminal proteins, LHBs, and middle MHBswere more sensitive to N-butyl DNJ than were small SHBs enrichedparticles.

Another of the advantages in the use of the defined N-alkyl derivativesof DNJ is their relative non-toxicity. For example, the N-butylderivative is known to be non-toxic (TD₅₀ >5 mM) at its effectiveconcentration for inhibition of HIV replication EC₅₀ =43 μM). See, e.g.,Bryant et al., Abstracts of 10th International Conference on AIDS,Berlin, Jun. 7-11, 1993. It is also shown herein that 90% of HepG 2.2.15cells infected with HBV and treated with 1000 μg/ml of the N-butylderivative of DNJ were as viable as untreated controls.

In view of the results demonstrated herein, it is believed that othercompounds that inhibit transport of HBV virions or steps in theglycosylation trimming pathway will be useful in inhibiting HBVmorphogenesis in tissue culture and mammalian hosts.

DETAILED DESCRIPTION OF THE INVENTION

While the specification concludes with claims particularly pointing outand distinctly claiming the subject matter regarded as forming theinvention, it is believed that the invention will be better understoodfrom the following illustrative detailed description taken inconjunction with the accompanying drawings in which:

FIG. 1 shows the genetic map of HBV envelope proteins. The upper-mostline shows a linear map of the HBs genes, with the preS1, preS2 and Sdomains indicated. Numbers under this map indicate the boundaries of thedomains, expressed as amino acid number. The numbers vary for differentHBV strains. The percentage values in the right-hand column show thefraction of non-, mono-, and di-glycosylated proteins for each HBs.Values are from Gerlich and Bruss, in Molecular Biology of HeDatitis BVirus, ed. A. McLachlan, CRC Press, pp. 109-144 (1992), and in HepatitisB Vaccines in Clinical Practice, ed. R. W. Ellis, Marcel Dekker, Inc.,pp. 41-82 (1992).

FIG. 2 in two parts, FIGS. 2A and 2B, shows autoradiograms of HBV DNA inthe media and cells of cultures treated with N-butyldeoxynojirimycin(NBDNJ). HepG 2.2.15 cells were grown for 6 days in the presence of theindicated concentration of NBDNJ with one change of medium. After thesixth day (seventh day in culture), cells and medium were collected.Autoradiograms of the viral DNA detected by hybridization of membranesto HBV probes are shown.

FIG. 2A: autoradiogram of a Southern blot of DNA recovered from themedium of 2.2.15 cells maintained in medium without NBDNJ (lanes 1 and2); and in the following NBDNJ concentrations: 200 μg/ml (lane 3); 500μg/ml (lane 4); and 1000 μg/ml (lane 5).

FIG. 2B: autoradiogram of a Southern blot of total intracellular DNA,digested to completion with EcoRI, from cells maintained in the absenceof NBDNJ (lane 1) and in the presence of the following NBDNJconcentrations:

lane 2: 200 μg/ml;

lane 3: 500 μg/ml;

lane 4: 1000 μg/ml.

Lane 5: EcoRI digested DNA isolated from virions prepared from untreated2.2.15 cells.

Lane 6: plasmid DNA as a hybridization control. Arrows: indicate theexpected mobility for relaxed circular HBV genomes (A) and linearized3.2 kb genomes (B).

FIG. 3 in four parts, FIGS. 3A, 3B, 3C and 3D, shows gel electrophoresisand histograms of HBV DNA in the cells and medium of HepG2 cellsinfected with HBV and treated with NBDNJ.

FIG. 3A and FIG. 3C: DNA from virions, immunoprecipitated withmonoclonal antibody to MHBs, was amplified by polymerase chain reaction(PCR) and resolved by agarose gel electrophoresis.

Lane 1: molecular weight markers;

Lane 2: Blank;

Lane 3: Medium from cells that received no NBDNJ;

Lanes 4 and 5: medium from cells that received 200 μg/ml NBDNJ;

Lanes 6 and 7: 500 μg/ml NBDNJ;

Lanes 8 and 9: 700 μg/ml NBDNJ, 10 and 11: 1000 μg/ml NBDNJ.

These bands were imaged by densitometry and the areas under the peaksare shown in FIG. 3C in which the average of the two samples of eachNBDNJ concentration was plotted.

FIG. 3B and FIG. 3D: DNA from the intracellular compartment of thecultures infected in FIG. 3A with HBV and treated with NBDNJ wereamplified by PCR. The lanes contain amplified DNA from the followingsamples:

Lanes 1 and 2: No NBDNJ;

Lanes 3 and 4: 200 μg/ml NBDNJ;

Lanes 5 and 6: 500 μg/ml NBDNJ;

Lanes 7 and 8: 700 μg/ml. NBDNJ;

Lanes 9 and 10: 1000 μg/ml. NBDNJ.

FIG. 3D: Histogram of the area under the averaged peaks of thedensitometric tracing of the gel in FIG. 3B. FIG. 4 in six parts, FIGS.4A, 4B, 4C, 4D, 4E and 4F, shows HBV antigens present in theunfractionated culture medium and partially purified virionpreparations. Equal volumes of the indicated samples were tested for HBVenvelope antigens using the enzyme-linked imminosorbent assay (ELISA)method. Samples of unfractionated medium (FIGS. 4A, 4B and 4C) orpartially purified virus (FIGS. 4D, 4E and 4F) from 3-day cultures weretested for HBV SHBs ("S) (FIGS. 4A and 4D), LHBs: "PreS1" (FIGS. 4B and4E) or MHBs "PreS2" (FIGS. 4C and 4F) epitopes. Cultures were maintainedin the indicated concentrations of NBDNJ, expressed in millimolar unitsand shown along the X axis.

For comparison, 2.28 mM approximately equals 500 μg/ml of NBDNJ. The Yaxis shows the calorimetric signal of the ELISA reaction, in arbitraryOD units, read by the plate reader.

FIG. 5 in three parts, FIGS. 5A, 5B and 5C, shows Western blot analysisof LHBs and MHBs in the medium of NBDNJ treated and untreated cultures.Polyethylene glycol (PEG) precipitates of culture medium (FIG. 5A) orpartly purified virions, HBs filaments and spheres from untreated (FIG.5B) and NBDNJ treated (FIG. 5C) cultures were resolved by SDS-PAGE(13.5% acrylamide), transferred to immobilin paper and incubated withPreS1 and PreS2 specific monoclonal antibody.

Lanes are as indicated at the top of each part of FIG. 5. 1.0 μg of HBVgenotype D, purified from human serum is used as a control.

Molecular weight markers (mw) in kilodaltons (kd) are shown at the rightside of each part of FIG. 5.

Arrows at the left show LHBs (S1) and MHBs (S2) polypeptides.

The HBV envelope contains three co-carboxy-terminal proteins (HBs),termed large (LHBs), middle (MHBs) and small (SHBs) protein (see FIG.1). These proteins result from the alternate translation initiation of asingle open reading frame (ORF) [Ganem, in Hepadnaviruses, eds. Masonand Seeger (Springer-Verlag), pp. 61-84 (1991)]. All three HBs proteinsoccur with complex type N-linked oligosaccharides at amino acid 146 ofthe S domain [see FIG. 1 and Gerlich and Bruss, in Molecular Biology ofHepatitis B Virus, ed. A. McLachlan, CRC Press, pp. 109-144 (1992), andin Hepatitis B Vaccines in Clinical Practice, ed. R. W. Ellis, MarcelDecker, Inc., pp. 41-82 (1992)].

MHBs (but never LHBs) also occurs with hybrid type oligosaccharideswithin the preS2 domain. During natural infection with HBV, the liverproduces a large excess of HBs proteins which are secreted as eitherfilamentous or spherical sub-viral particles of 20 nM in diameter[Ganem, in Hepadnaviruses, pp. 61-84 (1991); and Gerlich and Bruss, inMolecular Biology of Hepatitis B Virus, and in Hepatitis B Vaccines inClinical Practice, supra.] HBs spheres are most abundant and containfive to ten times less LHBs than do HBs filaments and HBV particles.MHBs is a minor component of all three types of particles [Gerlich andBruss, in Molecular Biology of Hepatitis B Virus, and in Hepatitis BVaccines in Clinical Practice, supra.]

The morphoaenesis of HBV is complex. Preassembled viral core particlesare believed to attach to the cytosolic sides of viral envelope(surface) proteins, which have inserted into the endoplasmic reticulum(ER) membrane [Gerlich and Bruss, in Molecular Biology of Hepatitis BVirus, and in Hepatitis B Vaccines in Clinical Practice, supra.]

After acquiring envelopes, virions bud to the lumen of the ER, fromwhere they are transported through the Golgi apparatus into theextracellular fluid. Immature glycoproteins contain three terminalglucose residues on the N-linked oligosaccharides.

The removal of terminal glucose residues is thought to play an importantrole in the migration of immature glycoproteins from the ER to the Golgi[Datema and Romero, Pharmacol. Thera. 33, 221-286 (1987)]. The iminosugar, NBDNJ, is a potent inhibitor of α-glucosidase I, a cellularenzyme which removes terminal glucose residues from nascentoligosaccharides, and has been found to suppress formation of cytotoxicHuman Immunodeficiency Virus (HIV) in vitro [Karpas et al, Proc. Natl.Acad. Sci. USA 85, 9229-9233 (1988); U.S. Pat. No. 4,849,430; and Walkeret al., Proc. Natl. Acad. Sci. USA 84, 8120-8124 (1987). Since HBVsecretion requires LHBs and SHBs, both of which bear N-linkedoligosaccharides, the effect of NBDNJ upon virus synthesis was tested.

In order to further illustrate the invention, the following detailedexamples were carried out although it will be understood that theinvention is not limited to these specific examples or the detailsdescribed therein.

EXAMPLES

METHODS

Cells and media:

HepG2 cells were purchased from the European Collection of Animal CellCultures (Porton Down, UK). HepG2 2.15 [2.2.15, Sells and Chen, Proc.Natl. Acad. Sci. USA 84, 1005-1009 (1987)] cells were obtained from Dr.George Acs (Mt. Sinai Medical College, New York, USA).

All tissue cultures were maintained in 5% C0₂ in RPMI 1640 (GIBCO)medium, supplemented with 10% heat inactivated fetal calf serum(Techgen, London, U.K.), 50 units/ml. of penicillin and streptomycin, 1mM glutamine (GIBCO). For 2.2.15 cells, 200 μgs/ml. Antibiotic G418(Genticin, GIBCO) was added to the medium, as in Sells and Chen, Proc.Natl. Acad. Sci. USA 84, 1005-1009 (1987).

Cell viability was measured by flow cytometry using a FACscan cytometer,Becton Dickinson, Sunnyvale, Calif., USA, after incubation withpropidium iodide, as in Platt and Jacob, Eur. J. Biochem. 208, 187-193(1992).

Infection of HepG2 cells:

HBV was purified from human serum or from the medium of cultured cellsby sedimentation to between 40 and 46% sucrose (w/w) followingultracentrifugation, [Seifer et al., Virol. 179, 300-311 (1990)].Virions were dialyzed in 0.02 M Potassium Phosphate Buffer, pH 7.4,concentrated, treated with V8 protease (from Staphylococcus aureus,Sigma Chemical Co.) overnight at 37° C. centrifuged for 8 hours througha 20% sucrose/0.01M Tris, pH 7.4, 0.14 M NaCl, 0.005 M EDTA (TNE)cushion. The pellets were re-suspended in growth medium and then used toinoculate HepG2 cells.

Iminosugar compounds.

The synthesis of NBDNJ is well-known and is described by Fleet et al,FEBS Letters 237, 128-132 (1988). NBDNJ was provided by G. D.Searle/Monsanto Co. as compound SC-48334.

Detection of viral DNA.

Medium from approximately 5×10⁶ cells was precipitated with polyethyleneglycol (PEG) 8000 (Sigma), after clarification, as in Sells and Chen,Proc. Natl. Acad. Sci. USA 84, 1005-1009 (1987), re-suspended in 0.5 ml.phosphate buffered saline (PBS) and sedimented through a cushion of 20%sucrose in PBS for 5 hours at 50,000 rpm in a Beckman T100.3 rotor(approx. 75,000×g).

DNA was prepared from the pellets as in Sells and Chen, Proc. Natl.Acad. Sci. USA 84, 1005-1009 (1987). For Southern blots [Maniatis etal., Molecular cloning, a laboratory manual, Cold Spring HarborLaboratory, Cold Spring Harbor, N.Y. (1982)], DNA was resolved byelectrophoresis through 1.2% agarose, transferred to H+ bond (Amersham)filter paper and hybridized with radioactive ³² P (Amersham) HBV probe(made by the random priming method described by the kit manufacturer,(Amersham), using pHBV as template [Foster et al., Proc. Natl. Acad.Sci. USA 88, 2888-2892 (1991)].

Progeny virus in the medium of HepG2 cells infected with serum derivedHBV was detected by precipitating medium with monoclonal antibody to thePreS2 epitopes. DNA from immunoprecipated virions was amplified by apolymerase chain reaction (PCR) using primers from nucleotides 2815 and190 with respect to the viral genome (using the EcoRI site as nucleotide1). DNA was prepared from cell lysates by standard methods as describedby [Maniatis et al., Molecular cloning, a laboratory manual, Cold SpringHarbor Laboratory, Cold Spring Harbor, N.Y. (1982)].

Detection of HBV proteins by the Enzyme Linked Immunoabsorbant assay(ELISA).

Monoclonal antibodies used herein are well-known and are described byHeerman et al., J. Virol. 52, 396-402 (1984), for antibody to PreS1 (MA18/7); Heerman et al., Intervirology 28: 14-21 (1987), for antibody toPreS2 (Q 19/10), see also Gerlich and Bruss, Molecular Biology ofHepatitis B Virus, ed. A. McLachlan, CRC Press, 109-144 (1992), and inHepatitis B Vaccines in Clinical Practice, ed. R. W. Ellis, MarcelDekker, Inc., pp. 41-82 (1992); and Heerman et al., in Viral Hepatitisand Liver Disease, ed. Zuckermann, A. R. Liss, 697-701 (1988), forantibody to S (C20-2).].

Samples were incubated in microtiter wells coated (overnight at 4° C.)with monoclonal antibodies specific for the LHBs, MHBs or SHBs epitopesand blocked with 1% BSA in PBS. After incubation with virus samples infor 1 hour (37° C.), plates were washed 4 times with PBS/0.1% Tween 20non-ionic detergent.

Bound antigen was detected by incubation with peroxidase conjugated goatanti-HBs antibody (Behring) followed by development inorthophenylenediamine (0.33 mg/ml PBS-peroxide solution). Opticaldensities were read in a Behring plate reader. Tests on purified virusand total medium were conducted over a series of sample dilutions toinsure proper quantification.

Western Blots:

Samples were dissolved in loading buffer, resolved by electrophoresisthrough 13.5% SDS polyacrylamide gels (SDS-PAGE) and transferred to PVDF(Millipore) membranes and blocked with 5% powdered milk, as in Gultekinand Heerman, Analytical Biochem. 172, 320-329 (1989). After incubationwith primary antibody overnight at room temperature in 1% bovine serumalbumin (BSA) in TNE and second antibody (peroxidase conjugated goatanti-mouse IgG serum) for 1 hour in TNE at room temperature, membraneswere developed in peroxide-diaminobenzidine (Sigma) PBS, as described inthe manufacturer's instructions.

RESULTS

NBDNJ reduces the amount of virion associated HBV DNA released into themedia by 2.2.15 cells:

2.2.15 cells are derived from the HepG2 line and chronically secreteinfectious HBV as well as sub-viral particles (HBs particles andspheres) into the culture medium [Heerman et al., J. Virol. 52, 396-402(1984); and Sells et al., Proc. Natl. Acad. Sci. USA 84, 1005-1009(1987)].

To determine the effect of NBDNJ upon the production and secretion ofHBV, 2.2.15 cells were maintained in medium containing a range of NBDNJconcentrations for six days, with one change of medium on the third day.After six days in compound, DNA was isolated from virus (as describedunder "Methods" above). Viral DNA was detected by hybridization ofSouthern blots to radioactively labeled HBV DNA probes, as shown in FIG.2A.

Virus-specific DNA, migrating with the expected mobility of relaxedcircles and linear genome length DNA [Sells et al., J Virol. 62,2836-2844 (1988)] was detected in the samples derived from untreatedculture medium (lanes 1 and 2). There is a clear dose-dependent decreasein virus specific DNA obtained from the media of cells treated withNBDNJ (lanes 3, 4 and 5). The autoradiograph shown in FIG. 2A wasquantified by densitometry. Densitometry revealed that 500 μg/ml (2.28mM) and 1000 μg/ml of NBDNJ resulted in decreases of 90 and 99%,respectively.

The decrease was not due to toxicity of NBDNJ, since 90% of the cellsmaintained in 1000 μgs/ml NBDNJ for six days were as viable as untreatedcontrols, as determined by FACs analysis of propidium iodide stainedcells and .sup.[35] -S methionine incorporation into proteins.

2.2.15 cells treated with NBDNJ contain elevated levels of intracellularHBV DNA.

The NBDNJ-mediated decrease in virion associated HBV DNA in the mediumcould have been due to a decrease in viral DNA synthesis. Alternatively,it could have been due to a post synthetic event such as virionassembly, transport or egress from the cell. To distinguish betweenthese possibilities, the amount of intracellular HBV specific DNA inuntreated and NBDNJ treated 2.2.15 cells was compared.

Total cellular DNA was prepared from treated and untreated cells anddigested to completion with EcoRI to linearize viral genomes. Near equalmicrogram amounts of digestion products (as determined by ethidiumbromide staining and hybridization to a cell specific probe) wereresolved by electrophoresis, Southern blotted and hybridized with theHBV specific probe (FIG. 2B). Unit length HBV genomes migrating as 3.2kb bands are detected in DNA derived from untreated 2.2.15 cells(lane 1) and virions isolated from control culture medium (lane 5).

Lanes 2, 3 and 4 contain DNA derived from cells treated with 200, 500and 1000 μg/ml. NBDNJ, respectively. There is a clear dose-dependentincrease in the amount of HBV DNA present in NBDNJ treated cells, ascompared with untreated cells. Densitometry of this autoradiogramsuggests that cells treated with 200, 500 and 1000 μgs/ml. of NBDNJexhibit an 1.7, 3.0, 5.1 fold increase in HBV copy abundance,respectively, when adjusted for loading variation, using hybridizationto a cellular MHC class III gene, G1, as a loading control.

HepG2 cells infected with HBV and treated with NBDNJ release lessprogeny virus.

2.2.15 cells are a useful system to study HBV production in a stablytransfected environment. However, HBV pregenome synthesis, in thesecells, may occur from integrated viral DNA templates and not covalentlyclosed circular viral DNA templates, as is thought to occur duringnatural infection [Sells et al., J. Virol. 62, 2836-2844 (1988)].Moreover, these cells produce naked core particles as well as a varietyof subgenomic viral DNA products which are released in to the medium[Sells et al., supra].

Therefore, HepG2 cells were infected with protease-modified HBV. Thenext day, culture medium was replaced with either control medium ormedium containing various concentrations of NBDNJ. Five days afterinfection, progeny virions were immunoprecipated with monoclonalantibody specific for the central portion of the PreS2 domain. HBVspecific DNA sequences were amplified by PCR using HBV specific primers.Products of the reaction were resolved by agarose gel electrophoresisand imaged after ethidium bromide staining (FIG. 3A). The 519 base pairproducts (arrow, FIG. 3A) were quantified by densitometry analysis andthe plot is shown in FIG. 3C. Although PCR may underestimate thedifferences between initial DNA concentrations in samples, it is evidentthat medium from cells infected with protease processed virus and posttreated with 700 μg/ml (3.2 mM) NBDNJ contain an order of magnitude lessviral DNA than do untreated samples. These results show that the NBDNJmediated decrease in HBV released into the media is not peculiar to the2.2.15 transfected cell system.

HepG2 cells infected with HBV and treated with NBDNJ contain increasedamounts of intracellular HBV DNA.

Since the culture medium of HBV infected HepG2 cells treated with NBDNJwas similar to 2.2.15 cells, with respect to the reduced amount ofvirion associated DNA, it was of interest to know if there was aconcomitant increase in viral DNA within the treated cells. Totalcellular DNA was prepared from samples corresponding to those presentedin FIGS. 3A and C. Intracellular HBV specific DNA was amplified usingPCR. Products of the reaction were resolved by agarose gelelectrophoresis and the ethidium bromide stained gel (FIG. 3B) wasanalyzed by densitometry (FIG. 3D). Clearly, HepG2 cells infected withHBV and post treated with NBDNJ accumulate greater amounts of viral DNAthan do untreated cells.

The culture medium of NBDNJ treated and untreated 2.2.15 cells containsimilar amounts of HBV envelope antigen.

2.2.15 cells secrete virions as well as sub-viral particles [Sells etal., J. Virol. 62, 2836-2844 (1988)]. The NBDNJ-mediated reduction ofvirion-associated DNA in the culture medium could be a reflection of ageneralized decrease in the secretion of all HBs containing particles.Alternatively, there may have been a selective diminution of the rarevirion particle with a relative sparing of the other form(s), which arein vast excess.

To distinguish between these possibilities, the amount and nature ofenvelope antigens in the culture medium was determined by both ELISA andWestern analysis. ELISA analysis of SHBs, MHBs and LHBs antigens presentin clarified culture medium is shown in FIGS. 4A, B and C. The resultsshow that there is no significant effect upon the total amount of SHBs(S) and LHBs (PreS1) antigens in the medium. There is a modest,dose-dependent decrease in the total amount of MHBs (PreS2) antigen inthe medium. This decrease is approximately 2.5 fold at the highest NBDNJconcentration (4.5 mM or approximately 1000 μg/ml.). These results wereconfirmed by Western blot analysis. FIG. 5A shows Western blots ofmedium from control cultures and those treated with 1000 μg/ml. Here itis shown that medium from NBDNJ treated (FIG. 5A, lane 3) and untreatedcultures (FIG. 5A, lane 2) contain similar amounts of MHBs (S2) and LHBs(S1) antigens.

Therefore, NBDNJ does not cause a generalized reduction in the amount ofSHBs and LHBs antigens in the culture medium. There is, however, atwo-fold decrease in the amount of MHBs antigen in the medium of 2.2.15cells treated with 1000 μg/ml NBDNJ, as determined by ELISA.

The culture medium of NBDNJ-treated 2.2.15 cells contains reducedamounts of HBV envelope antigens sedimenting as intact virions.

To determine the amount of HBs present in intact virions, medium fromthe indicated cultures was fractionated through sucrose gradients byultracentrifugation. Secreted virions derived from treated and untreatedcultures and sedimenting to 40-46% sucrose were concentrated and testedfor HBs proteins by ELISA. FIGS. 4D, 4E and 4F show the results. Allforms of HBs were easily detectable in samples containing virionsprepared from the medium of untreated 2.2.15 cells.

On the other hand, there were virtually no detectable HBs proteins insamples prepared from the medium of cells treated with 2.25 and 4.5 mM(approximately 500 and 1000 μg/ml, respectively, for comparison) NBDNJ.This suggests a reduced amount of intact virus in the medium of thesesamples.

The ELISA results were confirmed by Western blot analysis of fractionsfrom the sucrose gradient containing either intact virus, filaments orspheres (FIGS. 5B, from untreated cultures, and 5C, from NBDNJ treatedcultures). Equal volumes from fractions of the sucrose gradient wereresolved by SDS gel electrophoresis, transferred to membranes, incubatedwith antibody specific for LHBs (PresS1), imaged and then furtherincubated with antibody specific for MHBs (PreS2) epitopes. Partiallypurified HBV derived from human serum is presented in lanes 5 (FIGS. 5Band 5C) as a control.

Fractions distal to the virion containing fractions (near the bottom ofthe gradient) were resolved in lane 1 to show specificity of theantibody. Lanes 2, 3 and 4 (FIG. 5B and FIG. 5C) contain the intactvirus, HBs filament and sphere containing fractions (respectively), asdefined by sedimentation in sucrose and the presence (for virions) andabsence (for sub-viral particles) of viral DNA. There is a decrease inthe amount of all HBs proteins present in virions, filaments and spheresprepared from the medium of NBDNJ treated cultures (compare lanes 2, 3and 4 in FIGS. 5B and 5C). The decrease in MHBs (PreS2 epitope) isparticularly severe. The images shown in FIGS. 5B and 5C) werequantified by densitometry.

Densitometry analysis revealed that the decrease of LHBs in NBDNJtreated virion samples, compared to untreated samples, was approximately4 fold. The decrease in MHBs, in the same lanes (FIG. 5C, lane 2compared with FIG. 5B, lane 2) was 12 fold. It is noted that thegradient used to separate the various forms of HBs protein results infractions "enriched" for different forms. That is, the virion containingfractions are likely to also contain filaments and spheres. This maycause an underestimation of the effect of NBDNJ upon the release ofvirions, relative to spheres and filaments, as judged by immunologicalanalysis.

Nevertheless, the results of the Western blot analysis are consistentwith those of the ELISA, in that medium from NBDNJ treated culturescontains similar amounts of total HBs antigens but greatly reducedantigenic material in virion fractions.

The inhibitory compounds described herein also can be used foradministration to patients infected with HBV by conventional means,preferably in formulations with pharmaceutically acceptable diluents andcarriers. These compounds can be used in the free amine form or in theirsalt form. Pharmaceutically acceptable salt derivatives are illustrated,for example, by the HCl salt.

These inhibitory compounds also can be used in the form of pro-drugssuch as the 6-phosphorylated derivatives described in U.S. Pat. Nos.5,043,273 and 5,103,008, and the O-acylated derivatives such asdescribed, e.g., in U.S. Pat. Nos. 5,003,072; 5,144,037; and 5,221,746.A preferred such derivative is 1,5-(butylimino)-1,5-dideoxy-D-glucitol,tetrabutyrate.

The amount of the active compound to be administered must be aneffective amount, that is, an amount which is medically beneficial butdoes not present toxic effects which overweigh the advantages whichaccompany its use. It would be expected that the adult human dailydosage would normally range from about one to about 1000 milligrams ofthe active compound. The preferable route of administration is orally inthe form of capsules, tablets, syrups, elixirs and the like, althoughparenteral administration also can be used. Suitable formulations of theactive compound in pharmaceutically acceptable diluents and carriers intherapeutic dosage form can be prepared by reference to general texts inthe field such as, for example, Remington's Pharmaceutical Sciences, Ed.Arthur Osol, 16th ed., 1980, Mack Publishing Co., Easton, Pa., U.S.A.

Various other examples will be apparent to the person skilled in the artafter reading the present disclosure without departing from the spiritand scope of the invention. It is intended that all such other examplesbe included within the scope of the appended claims.

What is claimed is:
 1. A method for the treatment of hepatitis B virusinfections in an infected human host comprising administering to saidhost an N-alkyl derivative of 1,5-dideoxy-1,5-imino-D-glucitol in whichsaid alkyl group contains from 3 to 6 carbon atoms in an effectiveamount to inhibit replication and secretion of hepatitis B virusvirions.
 2. The method of claim 1 in which the alkyl group is butyl. 3.The method of claim 1 in which the effective inhibitory amount is fromabout one to about 1000 mg.